Comparison of Transhydrogenase and Pyridine NucleotideCytochrome c ReducÃ-aseActivities in Rat Liver
and NovikoiF Hepatoma*
BALTAZAR
RETNAFARJE!ANDVANR. POTTER
(HcArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis.)
In a recent discussion of the biochemical as
pects of tumor uniformity and heterogeneity,
it
was suggested
that TPNH'-cytochrome
c reductase was a strategically
important
oxidative
component that needed further study in tumors
(25). It is the object of this report to examine that
enzyme, together with the related enzymes, DPNcytochrome c reductase and transhydrogenase,
in
rat liver and in the Novikoff hepatoma. The latter
tumor was chosen because of its relation to rat
liver, which has been studied extensively in this
laboratory in young rats and in adult rats follow
ing partial hepatectomy,
and because of the
extensive data available on the Novikoff hepa
toma (16, 21, 32-34).2
It would be impossible to detail the many de
velopments that facilitated the present study, but
three are particularly
noteworthy:
first, the de
velopment
of the centrifugal
technics for cell
fractionation,
which in turn led to the studies by
Hogeboom and Schneider (10) and by DeDuve
et al. (4), showing that TPNH-cytochrome
c re
ductase activity occurs both in the mitochondria
and in the "microsome fraction"; second, the dis
covery of the enzyme called pyridine nucleotide
transhydrogenase
in Pseudomonas fluorescens by
Colowick et al. (3) and its demonstration
in animal
* This work was supported in part by a grant (No. C-646)
from the National Cancer Institute, National Institutes of
Health. A preliminary report was given at the Annual Meeting
of the American Association for Cancer Research and reported
in Proc. Am. Assoc. Cancer Research, 3:241, 1957.
t Rockefeller Foundation Fellow, 1955-1957; Present
address, Departamento de Fisiopatologia, Facultad de Medi
cina, Universidad de San Mareos, Lima, Perú.
1Abbreviations used : DPN and TPX = oxidized diphospho- and triphosphopyridine
nucelotides, respectively;
DPNH and TPNH = reduced diphospho- and triphospho
pyridine nucleotides, respectively; Mt = mitochondria; Me =
microsomes.
* We are indebted to Dr. Claude Allard for making available
valuable unpublished data.
Received for publication July 8, 1957.
tissues by Kaplan et al. (14); and, third, the
demonstration
of the mitochondrial
membrane
(29, 31) and the use of "depleted" mitochondria
(15, 17, 28).
MATERIALS
AND METHODS
Rats bearing Novikoff hepatoma were kindly
supplied by Dr. A. Novikoff, and his method of
transplanting
the tumor intraperitoneally
was
used (21). Normal male rats, 160-200 gm., were
obtained
from the Holtzman
Rat Company,
Madison, Wisconsin. After the rats were killed
by decapitation,
the required tissues were re
moved and placed in cold isotonic sucrose. All
subsequent
manipulations
were carried out at
0°C.
Preparation of homogenates and ceil fractions.—
Homogenates,
in water or isotonic sucrose, were
prepared
in a glass-to-glass
Potter-Elvehjem
homogenizer. Mitochondria
were prepared in a
model PR-1 International
refrigerated centrifuge
by fractionation
of a 10 per cent homogenate in
0.25 M sucrose by the method of Schneider and
Hogeboom (27). The fluffy layer that appears
after washing the mitochondria
was always de
canted from the well packed pellet and was never
used in a regular experiment except in a single
one, in which it had characteristics
intermediate
between those of mitochondria and microsomes as
far as the enzymes studied are concerned.
The "microsome fraction" was prepared from
the mitochondrial
supernatant
by centrifugation
at 105,000 X g for 50 minutes in a Spinco pre
parative ultracentrifuge.
Preparation of depleted mitochondria and microsomal vesicles.—Mitochondria
and microsomes
equivalent to 2.5 gm. of tissue were resuspended in
5 ml. of glass-distilled water and incubated at
room temperature
for 2 hours with constant and
gentle shaking in an Erlenmeyer flask. The cell
fractions were then sedimented by centrifugation
1112
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1957 American Association for Cancer Research.
REYNAFAEJEANDPOTTER—Transhydrogenasein Liver and Tumor
at 0°C. at the same speed and time as were used
for their preparation.
The pellets were resuspended in cold isotonic sucrose and used for the
enzyme assays. The supernatant
from the de
pleted mitochondria
was further centrifuged in
the Spinco at 105,000 X g for 50 minutes to re
move any possible microsomal
contamination.
This supernatant
(washing water), as well as that
from depleted microsomes, was then checked for
enzyme activity and protein content.
Protein determinations
were carried out by the
biuret reaction (8).
Reaction mixtures.—The standard reaction mixture con
tained the following final concentrations in a total volume of
3.0 ml.: 0.04 M nicotinamide; 0.033 M phosphate buffer, pH
7.2; 3.0 mg. oxidized cytochrome c; 3.3 X 10~4 M potassium
cyanide; and 350 Mg.of TPNH, DPN, DPNH, or combinations
thereof. In many cases isotonicity was achieved by addition of
the right amount of solid sucrose to the medium, but it was
shown that, with hypotonie reaction mixtures, the results were
essentially the same. The cell fractions were kept in cold
isotonic sucrose prior to use. The amount of tissue used was at
a level of 1-1.5 mg. of protein/reaction mixture. In the case of
liver mitochondria this was roughly equal to about 25 mg. of
fresh whole liver. However, the amount varied above and be
low this figure, depending on the rate of the reaction; but the
fractions were usually added hi a volume of 0.1 ml. of isotonic
sucrose. Nicotinamide was used in the reaction mixture to
prevent enzymatic inactivation of pyridine nucleotides by the
specific nucleosidases (19). The cyanide was added to inhibit
cytochrome oxidase, which is present in the mitochondrial
fraction and which evidently is present to a small extent in the
microsome fraction. No reduction of cytochrome c occurs in
the absence of cyanide in mitochondria, but a fairly rapid re
duction occurs when microsomes are used in the absence of
cyanide (Chart 1). The results at the highest level of cyanide
are believed to depend upon the time of exposure of the cyto
chrome c to cyanide, as shown earlier by Potter (25).
Assay procedure.—Assays were developed according to
technics based on those used by Potter (25) and Lockhart and
Potter in 1941 (18) for the study of the DPNH-cytochrome c
reducÃ-ase,except that TPNH was also used and the existence
of transhydrogenase was recognized. In general, all the experi
ments were done at room temperature, and the reduction of
cytochrome c was measured at a wave-length of 550 m^t. A
model B Beckman spectrophotometer was used, and the change
in absorbancy was read every 15 seconds for the fast reactions
and every minute for the slower ones. A conversion factor for
the Amólesof cytochrome c reduced for a given change in the
E value was found by standardization against a Model DU
Beckman spectrophotometer. With the use of a 13 X 100-mm.
calibrated colorimeter tube as the reacting cell for the Beck
man B, the change in the E value, when oxidized cytochrome c
was converted into the reduced form, was lower than the cor
responding value obtained in a Beckman DU with the proper
cell by a factor equal to 1.5, which was used to correct our
figures. Usually reactions were started by adding one of the
pyridine nucleotides.
Reduction of cytochrome c upon the addition of DPNH was
used as a measure of the DPNH-cytochrome c reducÃ-aseand
its rate as a measure of the activity. Similarly, the reduction
upon the addition of TPNH was interpreted as due to the
TPNH-cytochrome c reducÃ-ase.The increase in the rate of
cytochrome c reduction by TPNH, when DPN was already
present, was an expression of the transbydrogenase.
1113
Chemical materials.—DPN, DPNH, TPNH, and heart
cytochrome c were products of the Sigma Chemical Company.
Other materials were commercial products of reagent grade.
RESULTS
Comparison of normal liver with Novikojf hepatoma.—Chart 2 represents a typical experiment
comparing
the activity of normal liver mito
chondria and Novikoff hepatoma mitochondria in
promoting the reduction of cytochrome c upon the
addition of DPNH, TPNH, or the combination
of DPN and TPNH. In general, the amount of
tissue was equivalent to 25 mg. wet weight of
original tissue. However, in the case of liver
mitochondria with DPNH, the amount used was
0.5
MITOCHONDRIA
30,4
MICROSOMES
3.3x IO~4M CN"
E
S 0.31
U)
7.0 x
0.2
NO CN
O.I
NO CN
I
3
O
i
MINUTES
CHART 1.—Cytochrome c reduction by TPNH in liver
mitochondria and microsome fractions at different levels of
cyanide. Reaction mixtures contained the standard additions
with cyanide used at the indicated levels. Reactions were
started by addition of TPNH.
one-fifth as much because of the rapid rate; and
in the case of hepatoma with TPNH ±DPN, the
amount was increased up to as much 150 mg.
equivalent. It can be seen (Chart 2) that DPNHcytochrome c reducÃ-ase in liver mitochondria was
more active than that present in hepatoma mito
chondria,
which nevertheless
were sufficiently
active to give a vigorous reaction. In normal liver
mitochondria
there was a slow rate with TPNH
and a somewhat faster reaction when DPN was
added to bring transhydrogenase
into play. In the
hepatoma
mitochondria
neither
TPNH-cyto
chrome c reductase nor transhydrogenase
activity
was in evidence, as shown by results with
TPNH + DPN. Chart 3 shows the results with
the microsome fraction. In this case the DPNH
system was active in both liver and hepatoma. In
liver there was activity with TPNH
and no
stimulation
when DPN was added, which fact
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1957 American Association for Cancer Research.
1114
Cancer Research
suggested either a lack of transhydrogenase or
saturation with DPN. The former explanation
appears to be the correct one. With the microsome
fraction from hepatoma, there was no activity
with TPNH + DPN, which observation indicated
NOVIKOFF
HEPATOMA
Mt
cell fraction might have passed into another
fraction or might have been in a discarded frac
tion. However, the results with whole homogenate
support the results with mitochondria and with
the microsome fraction: DPNH-cytochrome c re
ductase was present in both liver and hepatoma,
but TPNH-cytochrome c reductase and transhy
drogenase were too weak to be demonstrated in the
hepatoma. It may be stated here that neither
reductase nor transhydrogenase
was demon
strable in the supernatant from the microsomal
fraction from liver or hepatoma in isotonic
sucrose.
Table 1 summarizes a series of experiments in
which TPN and DPN-cytochrome c reductase
were determined in whole homogenate and cell
fractions of normal liver and Novikoff hepatoma.
30
MINUTES
CHABT2.—Pyrimidine nucleotide-cytochrome c reductases
and transhydrogenase activity of rat liver and hepatoma
mitochondria. All reaction mixtures contained the standard
additions plus 25 mg. equivalent of tissue, except for liver
DPNH and tumor TPXH±DPN, in which 5 and 150 mg.
equivalents of mitochondria were used, respectively.
•¿5-LIVER Me.
NOVIKOFF
HEPATOMA Me.
•¿4o
IO
IO
p
o
1234
MINUTES
•¿3-
CHART4.—Cytochrome c reduction by DPNH, TPNH, or
DPN+TPNH in whole homogenates from liver and Novikoff
hepatoma. Reaction mixtures contain standard additions plus
one-fourth (about 0.25 mg. of protein) the regular amount of
tissue, except for tumor DPN+TPNH
in which 25 mg. of
tissue was used.
•¿2ITPNH+DPN,
It is evident that DPNH-cytochrome c reductase
activity in hepatoma was less than in liver, but
MINUTES
the more striking result was that in hepatoma the
CHART3.—Pyridine nucleotide-cytochrome c reductases
TPN-cytochrome c reductase activity was neg
and transhydrogenase activity of rat liver and hepatoma "mi
crosome fraction." All reaction mixtures contained the ligible, both in comparison with the TPN enzyme
standard additions plus 25 mg. equivalent of tissue, except for of liver and with the DPN enzyme of the hepa
liver DPNH and tumor TPNH + DPN, in which 5 and 150 mg. toma itself.
equivalents of microsome fraction were used, respectively.
To learn whether the lack of activity with
Novikoff hepatoma on TPNH + DPN was simply
absence or negligible amounts of transhydro
an artefact or the result of some sort of inhibitor
genase and TPNH-cytochrome c reductase.
present in hepatoma, whole tumor and liver homo
Results in Chart 4 were obtained from experi
genates were incubated together for 20 minutes at
ments with whole homogenates of liver and of room temperature in a reaction mixture contain
hepatoma. This experiment was done because it ing all ingredients but cytochrome c. The reac
seemed possible that activity missing from one tions were started by adding cytochrome c. Data
012301234
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1957 American Association for Cancer Research.
REYNAFARJEANDPOTTER—Transhydrogenasein Liver and Tumor
in Chart 5 show the results on TPNH-cytochrome
c reductase, which is almost absent in Novikoff
hepatoma. The activity in liver appeared intact
after a period of 20 minutes of incubation with
tumor homogenate, as compared with that of liver
alone incubated
for the same period of time.
Fluoride, ATP (5), and Co A (1) did not show any
effect on the TPN reductase
and transhydrogenase activity of mitochondria
from Novikoff
hepatoma.
Experiments
with "depleted" mitochondria and
microsomes.—Since the enzyme transhydrogenase
promotes
the following reversible
TPNH
+ DPN+ ;=i DPNH
reaction
1115
supernatant
fluid or wash water. It may also be
seen (Chart 66) that the activity of the super
natant enzyme with TPNH alone was not stimu
lated when DPN was added, and no activity was
observed with DPNH. Thus, this activity may be
considered due to TPNH cytochrome c reduction
per se. A similar experiment was carried out with
microsomes, because Palade (22, 23) has shown
that the microsome fraction yields vesicles that
behave as osmometers and swell in distilled water.
However, Chart 6c shows that microsomes thus
treated not only retained DPNH activity but
also TPNH activity; and no indication of trans
hydrogenase
was ¡found, since the activity on
TPNH would appear not to require DPN (Chart
(13) :
+ TPN+ ,
TABLE 1
TOTALANDSPECIFICACTIVITYOFDPN ANDTPN-CYTOCHROME
c REDUCTASES
IN WHOLE
HOMOGENATE
ANDCELLFRACTIONS
FROMNORMALRATLIVERANDNOVIKOFFHEPATOMA
The activity is expressed in terms of Amólescytochrome c reduced/min/gm of fresh tissue or fraction therefrom
or per gram of protein. Figures are averages of two to four experiments.
RATDPN-cyt.
creductaseactivityPer
FRESHCELL
FRACTIONS
gm.protein
gm.
Per
tissue*
c.reductaseactivityPer
gm.protein
gm.
Per
tissue*
NOVIKOFF
HEPATOUA
DPN-cyt. c
TPX-cyt. c,
reductase
reductase
activity
activity
Per gm.
Per gm.
Per gm.
Per gm.
protein
tissue
protein
tissue
9.4
O.lf
04.2
0.24t
0.02f
5.7
41.4
3.2
0.42J
O.OSf
59.6278.3
5.516.4
Homogenate
Mitochondria
32.3208.5
2.221.7
Microsomes
21.3LIVERTPN-cyt.
2.6
* The biuret method for protein gave questionable results with whole homogenates.
t Values for hepatoma TPN-cytochrome c reduction are at the margin of precision for the present method.
its demonstration,
by the present methods, de
pends upon the availability of DPN in the cell
fraction studied, when TPNH is the hydrogen
donor. The liberation or "depletion"
of endoge
nous nucleotides including DPN from mitochondria
may be accomplished by different procedures (15,
17,28). The one we describe under "Methods" was
the most satisfactory for our purposes.
In Chart 2 it was shown that liver mitochondria
showed TPNH-cytochrome
c reductase activity in
the absence of added DPN, and it was thought
that if this result were due to intramitochondrial
DPN the activity would disappear if the mito
chondria were depleted. Chart 6a shows that this
result was obtained:
there was essentially
no
TPNH-cytochrome
c reductase activity in the
absence of added DPN; but, when DPN was
added with TPNH, cytochrome c reduction was
restored (Chart 6a). This does not necessarily
mean that mitochondria
lack TPNH-cytochrome
c reductase and that the TPNH activity seen in
fresh mitochondria
(Chart 2) is exclusively due
to intramitochondrial
DPN, because Chart 60
shows that the TPNH activity lost when mito
chondria were depleted could be recovered in the
•¿
LIVER
o—o LIVER4
HEPATOMA.<^X*''^
=1EoIOIOûo0.5-0.40.30.2O.I-0•
/&'/s/''V01
2345MINUTES
CHART 5.—TPNH-cytochrome c reductase activity in
whole liver homogenate alone, and in a mixture of whole liver
and tumor homogenates; 10 mg. of liver or 20 mg. of an equa
mixture of liver and tumor homogenates were incubated for 20
minutes at room temperature in 2.8 ml. of reaction mixture
containing all ingredients but cytochrome c. Reactions were
started by adding 0.2 ml. of a solution containing 3 mg. of
oxidized cytochrome c.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1957 American Association for Cancer Research.
1116
Cancer Research
6c). Chart 6d indicates that the microsomal fluid
or wash water did not have any considerable
activity.
From experiments of the type reported in
Chart 6, specific activity for each one of the
enzymes was calculated and summarized in
Table 2.
CHAHT6.—Pyridine nucleotide-cytochrome c reductases
and transhydrogenase activity in depleted liver mitochondria
(a) and microsome (e) fractions and their wash waters (6 and
d, respectively). Reaction mixtures contain the standard addi
tions plus the following amount of tissue protein: mitochondria
DPNH system = 0.28 mg.; mitochondria TPNH±DPN sys
tem =1.4 mg.; mitochondrial wash water = 0.75 mg.; microsome DPNH system = 0.3 mg.; microsome TPNH + DPN
system = 1.3 mg.; microsomal wash water=1.0 mg. protein.
In those experiments carried out with fresh
liver mitochondria, the increase in the rate of cy tochrome c reduction by TPNH in the presence of
added DPN when endogenous DPN was also
present was used merely as an evidence of trans
hydrogenase activity. However, it was not possible
to calculate its specific activity from that kind of
data, since, under the conditions employed,
transhydrogenase and TPNH-cytochrome
c reductase are performing cytochrome c reduction
simultaneously, and we do not know to what ex
tent each enzyme contributes to the rate of such a
reduction. Therefore, it would be erroneous to
calculate specific activity of transhydrogenase by
taking either the over-all reduction (DPN +
TPNH), or the difference between this over-all
reduction and that due to TPNH-cytochrome c
reductase (TPNH alone). The use of depleted
mitochondria obviates this difficulty, because
during depletion TPNH-cytochrome c reductase
is solubilized into the wash water (Chart 66 and
Table 2), leaving transhydrogenase attached to
the nonsoluble part of the mitochondria (Chart 6a
and Table 2), thus making it possible to calculate
its specific activity without interference.
In microsomes, where the process of depletion
failed to separate TPNH-cytochrome c reductase,
and in soluble fractions (wash water in Table 2)
no evidence of transhydrogenase activity was ob
tained, because it was demonstrated that the rate
of cytochrome c reduction with TPNH was not
TABLE 2
SPECIFICACTIVITYOFPYHIDINENDCLEOTIDE-CYTOCHKOME
c REDUCTASES
ANDTRANSHYDROGENASE
IN DEPLETEDCELLFRACTIONS
ANDTHEIR
WASHWATERSFROMNORMALLIVERANDNOVIKOFF
HEPATOMA
The data are expressed in terms of /»molescytochrome c reduced per minute
per gram of protein. Figures are averages of two to four experiments.
Liver
Depleted cell
fraction«
activity /gm
protein
Novikoff hepatoma
activity /gin
protein
0.3*
35.1
Mitochondria
0.0
0.0
Mit. wash water
0.0
0.0
Microsomes
0.0
0.0
Me. wash water
0.4*
1.5
Mitochondria
TPN-cyt. c
0.1
45. 6f
Mit. wash water
20.5
0.4
Microsomes
reductase
Me. wash water
2.1
0.3
Mitochondria
351. Of
112.2
DPN -cyt. c re3.4
Mit. wash water
2.5
39.5
200.0
Microsomes
ductase
Me. wash water
1.8
0.9
* Values for hepatoma transhydrogenase and TPNH -cytochrome c reductase
are at the margin of precision for the present method.
t In an experiment designed to test the rektion between the activities in fresh
and depleted mitochondria, 95 per cent of the original DPNH-cytochrome c re
ductase was recovered in the depleted mitochondria, and 67 per cent of the original
apparent TPNH-cytochrome c reductase was recovered in the mitochondrial
wash water. Cf. Chart 6a and 66.
Transhydrogenäse
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REYNAFARJEANDPOTTER—Transhydrogenasein Liver and Tumor
affected by DPN (Chart 66 and 6c). Measure
ments on endogenous DPN in the "depleted"
microsome fraction have not been made, but it is
assumed that none is present on the basis of the
results with depleted mitochondria (Chart 6a).
Experiments with irradiated, regenerating, and
newborn rat livers.—Sinceit is believed that the
enzymes here studied might play an important
role in many physiological as well as pathological
processes, exploratory experiments were carried
out to see if these enzymes show any change
under different conditions. Results with a pool of
livers from a litter of 7-day-old rats, as well as
with 12-, 18- or 24-hour regenerating livers, did
not show any gross variation from that described
for adult normal rat liver. Also, one experiment
with a 200-gm. rat, irradiated with 1500 r (20
roentgens/min) and killed 6 hours later, showed
results not significantly different from the normal
control.
Experiments with other tissues.—Preliminary ex
periments with certain other normal tissues, such
as rat heart and brain and guinea pig spleen and
pancreas, have been carried out. All three enzymes
studied were found in these tissues in different
amounts and with characteristic patterns of intracellular distribution for each one. No data com
parable to the hepatoma data were obtained thus
far, although negligible or negative values have
been reported (12) for transhydrogenase in cer
tain normal tissues. On the other hand, HeLa
cells, Yoshida ascites cells, primary rat hepatoma,
and mouse 129 hepatoma showed results markedly
similar to the hepatoma results, which might be in
agreement with the fact that TPN in tumors is
present at very low levels and almost exclusively
in the reduced form (7). A pooled sample of cul
tured human liver cells (Chang line), maintained
by Dr. D. L. Walker, was provided to us. The
assay results with whole homogenate resembled
the data with hepatoma and not the data with rat
liver, thus showing a tumoriike behavior in this
respect (24).
1117
this enzyme, expressed per gram of tissue, are
somewhat higher than values obtained by McIlwain and Tresize (20) and by Palade and Siekevitz (22), but lower than those reported by
Hogeboom (9). Transhydrogenase appears to be
limited to the mitochondria, being absent from the
microsome and soluble fractions of the cell. TPNcytochrome c reducÃ-ase,like DPNH-cytochrome c
reducÃ-ase, is present in both mitochondria and
microsome fractions, with a pattern again some
what different from that described by other work
ers (4, 10).
The studies with depleted mitochondria suggest
that DPNH-cytochrome c reductase and trans
hydrogenase are firmly attached to the nonsoluble
part of the mitochondria, which might well be the
"mitochondrial membrane" studied by Siekevitz
and Watson (29) and by Ball and Cooper (2),
whereas the mitochondrial TPNH system is
readily dissociated with water and passes into a
form not sedimented at speeds that ordinarily
bring down microsomes. Furthermore, it appears
not to be microsomal in origin, because it has a
negligible DPNH-cytochrome c reductase activ
ity. The absence of the latter enzyme requires that
the TPNH activity observed must be a true
TPNH-cytochrome c reductase and not an arte
fact due to transhydrogenase, and must also be
different from that of the microsome fraction,
since the enzyme from this fraction is not dis
sociated with distilled water by the present meth
od. However, much additional work will have to
be done to learn the distribution of the enzymes in
the various cell fractions and their role in oxidative
phosphorylation (15). The apparent lack of either
the transhydrogenase or the true TPNH-cyto
chrome c reductase in the Novikoff hepatoma
must of course be considered as a possible arte
fact, and further work needs to be done. Similarly,
the question of an absolute lack must be raised
with the physiological implication pointed out by
Potter in 1956 (26). Our studies with a few other
normal and tumor tissues should not be taken to
indicate that the combined deficiencies of TPNHDISCUSSION
cytochrome c reductase and transhydrogenase
From the results presented in this paper it ap
activity are unique characteristics of tumors thus
pears that normal rat liver contains all three of the fulfilling the requirements for the fundamental
enzymes studied, to an extent easily demonstrable
alteration that distinguishes cancer tissue from
by the present method. DPNH-cytochrome c re- normal tissue (26). Results obtained by Glock and
ductase occurs in both mitochondria and the McLean (7) showing that hepatoma and other
microsome fraction, although the pattern of intra- tumors examined contain low levels of TPN
cellular distribution of this enzyme appears differ might favor this conclusion. However, it appears
that the deficiencies of TPNH-cytochrome c re
ent from that reported by Hogeboom and Schneid
ductase and transhydrogenase are not exclusive
er (9, 11) and DeDuve et al. (4) in that mito
chondria are the site of the major activity of characteristics of neoplastic tissues, since Hum
DPNH-cytochrome c reducÃ-ase. Our values for phrey (12) reported negligible and negative values
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1957 American Association for Cancer Research.
1118
Cancer Research
of transhydrogenase for certain normal tissues not
yet studied in this laboratory. Also, a possible
indication that it may occur in some tumors
might be inferred by the report of Emmelot and
Brombacher (6), who found that thyroxine un
coupled oxidative phosphorylation in some tumor
mitochondria and not in others, and it appears by
the work of Ball and Cooper (2) that the thyrox
ine acted by inhibiting the transhydrogenase in
a preparation believed to be mitochondrial mem
branes; thus, a tissue lacking the enzyme should
accordingly not be affected by thyroxine. Data
presented by Waravdekar et al. (30) showed, inci
dentally, a significant TPNH-cytochrome c reductase activity in whole homogenate of Sarcoma
37, and Emmelot (personal communication) has
found the same enzyme in the microsome fraction
from certain tumors although at low levels.
It would be premature to discuss extensively
the implications of these findings until additional
work is carried out in a variety of tumors and
normal tissues. Nevertheless, it would appear to
be worth while to explore the physiological conse
quences of the enzymatic defect described in
hepatoma and to relate it to the excellent work
being done in other laboratories on the same
tumor.
SUMMARY
1. Pyridine nucleotide-cytochrome c reductases
and transhydrogenase have been examined in
whole homogenate and in cell fractions of normal
rat liver and Novikoff hepatoma.
2. DPNH-cytochrome c reductase was present
in mitochondria and microsome fractions from
both liver and hepatoma.
3. In liver the enzyme transhydrogenase was
limited to the mitochondria and was firmly at
tached to the nonsoluble part of this cell fraction.
It was absent from the microsome and soluble
fractions.
4. Transhydrogenase was absent or present
only to a negligible extent in whole homogenate
and cell fractions from Novikoff hepatoma, under
the conditions employed thus far.
5. There was a true TPNH-cytochrome c re
ductase in mitochondria and microsomes from
normal liver.
6. TPNH-cytochrome c reductase from liver
was firmly associated with microsomes but was
readily dissociated from mitochondria, passing
into a form that was not sedimented at a speed
of 105,000 X g. This soluble fraction did not con
tain DPNH-cytochrome c reductase.
7. TPNH-cytochrome c reductase activity was
absent or present in negligible amounts in whole
homogenate and cell fractions of Novikoff bepatoma, as determined by the present method.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Dr. A.
Novikoff for sending to us the Xovikoff hepatoma, to Dr. J. A.
Miller for supplying primary hepatoma, to Mr. R. Rueckert
for Yoshida ascites cells and HeLa cells, to Dr. D. L. Walker
for a pooled sample of cultured liver cells, and to Dr. A. J.
Dalton for mice bearing the 129 hepatoma.
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Comparison of Transhydrogenase and Pyridine
Nucleotide-Cytochrome c Reductase Activities in Rat Liver and
Novikoff Hepatoma
Baltazar Reynafarje and Van R. Potter
Cancer Res 1957;17:1112-1119.
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